Microencapsulated DNA for vaccination and gene therapy

ABSTRACT

A microparticle contains DNA coding for a polypeptide and oral administration of the microparticle leads to its expression. DNA coding for an immunogen is for stimulating antibody formation in a recipient and DNA coding for a non-immunogenic polypeptide is for gene therapy applications. DNA is incorporated into the microparticle without destruction of its function.

FIELD OF INVENTION

The present invention relates to microencapsulated DNA, to vaccinescomprising microencapsulated DNA, to methods of vaccination and tomethods of gene therapy comprising administration of DNA inmicroparticles, to methods of preparing microparticles containing DNAand to dried compositions comprising DNA-containing particles.

TECHNOLOGY REVIEW

The biodegradable polymer poly (DL-lactide-co-glycolide) (PLG) has beenused for many years by the pharmaceutical industry to deliver drugs andbiologicals in microparticulate form in vivo. The United States FDA hasrecently approved a PLG microsphere 30-day delivery system forleuprolide acetate (Lupran Depot (registered trade mark)) to be used inthe treatment of prostate cancer. A useful review of the potential ofpolymer microencapsulation technology for vaccine use is found inVaccine, 1994, volume 12, number 1, pages 5-11, by William Morris et al.

As an alternative to encapsulation, it is also known to deliver antigensin phospholipid vesicles called liposomes, as described for example byEppstein, D. A et al in Crit. Rev. Ther. Drug Carrier Syst. 1988, 5(2),pages 99-139. It is reported that a number of antigens have beendelivered intraperitoneally using liposomes, including cholera toxin,malaria sporozoite protein and tetanus toxoid, and that influenzaantigen has been delivered intra-nasally.

It is also known that, in certain circumstances, injection of naked DNAinto tissue can lead to expression of a gene product coded by that DNA.For example, in 1984, work at the United States NIH reported thatintrahepatic injection of naked, cloned plasmid DNA for squirrelhepatitis produced both viral infection and the formation of anti-viralantibodies in the squirrels.

WO-A-95/05853 describes methods, compositions and devices foradministration of naked polynucleotides which encode biologically activepeptides. This published application describes, inter alia, theinjection of naked DNA coding for an immunogenic antigen with the aim ofraising antibodies in the recipient of the naked DNA.

Liposomal delivery of DNA is also known, and is described, for example,in EP-A-0475178.

An alternative method for obtaining expression of a desired gene productis described in EP-A-0161640, in which mouse cells expressing bovinegrowth hormone are encapsulated and implanted into a cow to increasemilk production therein.

EP-A-0248531 describes encapsulating linear poly (I:C) in microcapsulesand using these to induce production of interferon.

WO-A-94/23738 purports to describe a microparticle containing DNA incombination with a conjugate that facilitates and targets cellularuptake of the DNA. In working examples, bombardment of cells bymicroparticles containing Tungsten is described. These examples appearlittle different to conventional bombardment of cells with DNA-coatedmetal particles. Furthermore, sonication is proposed in microparticlemanufacture, a step that is known to risk DNA damage but the presenteddata is inadequate and inappropriate to determine the integrity of theencapsulated DNA.

SUMMARY OF THE INVENTION

In the present invention, it is desired to deliver, in vivo, DNAencoding proteins with immunogenic, enzymatic or other useful biologicalactivity, usually under the control of an active eukaryotic promoter.Objects of the invention include improvement on vaccination therapiesknown in the art and improvement upon prior art gene therapy methods.

Improvement of or alternatives to existing compositions and methods aredesirable as these existing methods are known to contain a number ofdrawbacks.

WO-A-95/05853 describes administration of naked polynucleotides whichcode for desired gene products. However, the compositions and methods inthis publication are suitable only for injection, requiring sterileprocedures, being in itself an unpleasant and awkward route ofadministration.

WO-A-94/23738 purports to provide a process in which encapsulated DNA isreleased from the particles in the body of the recipient and then takenup by cells, although no accomplished in vivo examples are presented.

The invention seeks to provide novel compositions and methods ofadministration thereof that improve upon existing vaccination and genetherapy techniques and are effective in vivo, or at least overcome someof the problems or disadvantages identified in known compositions andmethods.

It is known that DNA is readily damaged so that it is no longer capableof inducing expression of a gene product. Surprisingly, the inventorshave succeeded in devising a technique for encapsulation of DNA withinpolymer particles, such that the DNA retains sufficient integrity toinduce expression of a gene product coded thereby. The inventors havealso succeeded in devising a DNA-containing microparticle suitable formammalian vaccination or for gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the components of a protein-expressingplasmid suitable for incorporation into a DNA-containing capsuleaccording to the invention;

FIG. 2 illustrates the results of Example 3, namely induction ofluciferase-specific serum antibodies by PLG-encapsulated plasmid DNA,and in which the left column indicates antibody titre after 3 weeks withthe right column showing antibody titre after 6 weeks (i.m. representsintra muscular, i.p. represents intra peritoneal, the bottom portion ofeach column represents IgG levels, the top portion resents IgM levels;

FIG. 3 shows dose-response data for injected and oral doses ofencapsulated DNA, with the titre of IgG, IgM and IgA in each case;

FIG. 4 shows: A—agarose gel dectrophoresis of plasmid DNA followinghomogenisation in a Silverson mixer at 2000 and 8000 rpm for 0-300seconds; and B—agarose gel dectrophoresis of DNA before and afterencapsulation; and

FIG. 5 shows stool IgA response to DNA within PLG microparticals.

FIG. 6 shows the results of oral administration of PLG-encapsulated DNAexpressing measles virus N protein; and

FIG. 7 shows the results of oral administration of PLG-encapsulated DNAexpressing rotavirus VP6 gene: A—faecal rotavirus—specific IgA response,B—rotavirus shedding after challenge of orally immunized mice.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, a first aspect of the invention provides a pharmaceuticalcomposition comprising DNA encapsulated in a polymer, said DNAcomprising a sequence coding for a polypeptide and wherein thecomposition is adapted to induce expression in a recipient of the codingsequence. Preferably, the coding sequence is accompanied by a promoterpromoting expression of the sequence. Where the pharmaceuticalcomposition is for use on mammals, it is convenient to use a eukaryoticpromoter and especially a promoter that operates in a wide variety oftissue types. In particular embodiments of the invention, a tissue—orcell type—specific promoter is used.

In use, the pharmaceutical composition is orally administered, and thecoding sequence is expressed leading to desired therapeutic effects.

A composition of the invention suitable for vaccination contains asequence coding for an immunogen. Following administration of thecomposition, expressed immunogen elicits production of antibodies withinthe recipient, thereby contributing to vaccination of the recipient.

A specific embodiment of the invention, described in an example below,contains a DNA sequence coding for a protein. It has been administeredin vivo and has been found to induce expression in a mammal of thatprotein. The expression was detected by measurement of production ofantibodies specific for that protein. The composition of the specificembodiment comprises microparticles in the size range up to about 10 μm.

Generally, the microparticles of the invention are intended to entercells of the recipient by phagocytosis, for example phagocytosis bymacrophages or other antigen presenting cells. Subsequently, the body ofthe microparticle breaks down in the intracellular space and the DNA isreleased. It is preferred that the microparticles of the invention arein the size range 0.01 μm to 30 μm, with 1 μm to 10 μm being a morepreferred range. These sizes have been found to be suitable for reliablyachieving in vivo expression of the DNA. It is also to be noted thatagents promoting uptake of the DNA are not needed in microparticles ofthe invention—as the microparticle size determines its uptake.

An alternative composition, according to the invention, contains asequence coding for a non-immunogenic product. Such a composition isparticularly useful for gene therapy, wherein it is desired to expressin a recipient a gene product that is non-expressed, or expressed at alevel that it is desired to increase. In this case, a composition of theinvention comprises a DNA sequence coding for a desired product, andwherein following administration of the composition expression of thecoding sequence results in an increased level of the desired product,giving the gene therapy effect.

It is again a feature of the invention that microparticles for genetherapy applications have sizes in the range 0.01 μm to 30 μm,preferably 1 μm to 10 μm, to target their uptake by phagocytosis.

Specific embodiments of the invention, for use in vaccination, comprisea DNA sequence coding for a protein or an immunogenic component thereof,or an immunogenic fragment or variant thereof, of a virus, bacterium orother pathogenic microorganism. The protein is, for example, an HIVprotein, an influenza virus protein, a measles virus protein, ahepatitis virus protein (such as hepatitis surface antigen) or apertussis protein. Further, where the composition is for oral use, itcan conveniently also contain a taste-enhancing agent. The term“taste-enhancing agent” is intended to encompass sweeteners, flavouringsand agents that mask any unpleasant taste from other components of thecomposition. It can conveniently be enterically coated orco-administered with an appropriate antacid formulation.

In a specific embodiment described below, a preparation ofmicroparticles contains a DNA sequence coding for a measles protein.Oral administration of the microparticles elicited an increase inantibodies specific for that protein. Likewise, another microparticlepreparation contains a DNA sequence coding for a rotavirus protein. Oraladministration of these microparticles preparation elicitedanti-rotavirus protein antibodies and a protective effect againstchallenge by the virus.

Specific embodiments of the invention, suitable for gene therapy,comprise a DNA sequence coding for an enzyme or another protein neededfor the treatment of genetic disease. For example, a DNA sequence codingfor glucocerebrosidase is suitable for the treatment of Gaucher'sdisease.

The inventors have thus provided DNA encapsulated within a polymer suchthat the ability of DNA to code for a desired gene product issubstantially not affected by the encapsulation process. It is knownthat DNA can readily be damaged by emulsifying and other steps necessaryfor production of polymer particles. The inventors have provided forencapsulation of DNA such that sufficient operative DNA is encapsulatedfor a biological effect to be obtainable upon administration of theencapsulated DNA.

The invention offers advantages, in that encapsulated DNA is suitablefor oral administration, avoiding the unpleasant and awkward aspectsassociated with having to inject DNA preparations described in the priorart. Specific embodiments in examples described below have beensuccessful in inducing immunogen-specific antibodies in response to oraladministration of a composition of the invention. In addition, theencapsulated DNA formulation is suitable for drying, e.g. freeze drying,into a form that is stable over long periods and is suitable forstorage. Further, for many vaccine applications it would be advantageousif, as well as a systemic humoral and cell—mediated immune response,immunity at mucosal surfaces could also be evoked. Specific embodimentsof the invention, described below, have been demonstrated to elicitsignificant increases in specific IgA antibodies, following oraladministration. The invention thus provides a pharmaceutical compositioncomprising DNA within a polymer particle, the DNA encoding apolypeptide, and the composition being adapted to induce mucosalpolypeptide specific IgA antibodies in a recipient.

The polymer of the microparticle of the invention preferably is bothbiodegradable and non-toxic. Suitable polymers include lactidecontaining polymers and glycolide containing polymers and copolymers of0-100:100-0 lactide:glycolide. In a specific embodiment of theinvention, the polymer comprises poly (DL-lactide-co-glycolide),otherwise referred to as PLG, chosen as it has been approved for humanand veterinary use.

The products of the invention are typically for in vivo use on animals,in particular animals. The polymer of the microparticle should thereforebe non toxic in vivo and suitable for pharmaceutical use. The polymershould further be biodegradable—either by consisting of or comprisingbiodegradable polymer—so that it releases its DNA in the recipient.There exists in the art an extensive literature on polymers suitable forhuman and animal oral use and a person of skill in the art will be ableto adapt polymers of the art into the microparticles of the presentinvention without difficulty. In this connection, the disclosures ofEP-A-0451390, WO-A-95/31184 and WO-A-95/31187 are incorporated herein byreference.

The DNA contained within the particle will typically comprise doublestranded DNA. The construction of a suitable DNA sequence for use in theinvention will be appreciated by persons of skill in the art. It ispreferred that the sequence comprises both a transcriptional promoterand a gene coding sequence. It is further preferred that the DNAsequence provides for a transcription termination and polyadenylationdownstream of the coding sequence.

It is particularly preferred that the DNA be double stranded, circularand super coiled. It has been observed that during manufacture ofparticles the DNA is subjected to severe shear forces. Using particularmild particle manufacturing conditions, the inventors have managed toretain functional DNA, though have observed that previously supercoiledDNA may become partly converted to the open circular form in theprocess.

Plasmid DNA is particularly suitable and is used in the specificembodiments of the invention described below. As there is extensiveliterature relating to plasmid manufacture a person of skill in the artwill readily be able to prepared a plasmid suitable for themicroparticle of the invention. In general, plasmids incorporating anyeukaryotic promoter sequence are suitable.

A further optional feature of the invention is that DNA—containingpolymer particles can be manufactured so as to have different half-livesin vivo. When administering an antigen during vaccination, it may beadvantageous for the antigen to be delivered over as long a time frameas possible. A particular embodiment of the invention provides a vaccinecomprising first and second vaccine components, the first vaccinecomponent comprising polymer-encapsulated DNA wherein the DNA includes asequence coding for an immunogen and wherein the polymer has a firsthalf life in vivo, and a second vaccine component comprisingpolymer—encapsulated DNA, wherein the DNA contains a sequence coding foran immunogen and wherein the polymer has a second half-life in vivo. Therespective half-lives could be up to 5 days and more than 5 days. In oneexample, the immunogen of the first and second vaccine components arethe same. Alternatively, the respective vaccine components can containDNA sequences coding for different immunogens.

In an embodiment of the invention, the half-lives of the respectivefirst and second vaccine components are up to two days, and more thantwo weeks. In a further embodiment, the first and second half-livesdiffer by at least an order of magnitude.

In use of a specific embodiment of the invention, described in anexample below, a plasmid encoding luciferase is under control of thehuman cytomegalovirus immediate early promoter and is encapsulatedwithin PLG in particles around two μm in size. This encapsulated DNA wasadministered to mice and elicited anti-luciferase antibodies that weredetected over a period of several weeks. The production of antibodies inresponse to encapsulated DNA according to the invention was comparedwith antibody production in response to administration of naked DNAintraperitoneally and orally. In both cases, encapsulated DNA elicitedequivalent or significantly higher amounts of IgG and IgM, and alsoevoked a significant IgA response.

A second aspect of the invention provides a method of encapsulating DNAin a polymer such that biological activity of the DNA is retained to asignificant extent. In an embodiment of the second aspect, a method forencapsulating DNA within a polymer particle, said DNA being capable ofinducing expression of a coding sequence within said DNA, comprisespreparing a (water-in-oil)-in-water emulsion to form microparticles andseparating subsequently produced DNA-containing microparticles bycentrifugation. Resultant microparticles preferably have sizes in therange 0.01 μm to 30 μm, more preferably 1 μm to 10 μm.

The method of the invention is carried out under conditions that ensureat least a portion of the DNA is not damaged during manufacture of theparticles and thereby retains its ability to induce expression of itsgene coding sequence.

It is essential that DNA is incorporated into the microparticles, andDNA incorporation is increased by preparing a solution of DNA plus analcohol, adding microparticle polymer and forming microparticlestherefrom. The alcohol content of the solution suitably varies between1% and 60% and preferably between 5% and 40%. In specific embodiments ofthe invention the alcohol content is around 15-35%, more particularly20-30% for microparticles made from PLG, producing DNA incorporation of25% and above, up to 50-60%. Ethanol is particularly suitable; methanoland propanol and other alcohols that do not denature DNA are alsosuitable, and the alcohol is preferably a straight chain or branchedC₂-C₁₀ alcohol.

It is also preferred that the emulsification step or steps of the methodbe carried out under conditions of reduced shear stress, and this isoptionally achieved by use of an emulsifying speed that is sufficient toobtain an emulsion and to form microparticles in the desired size rangebut not so high that all DNA is damaged by excessive shear. In anembodiment of the invention described below the emulsifying mixer speedis modified so that at least 25% DNA activity (assayed by transformationof competent bacteria or transfection of cultured cells) is retained inthe resultant microparticles that contain DNA. Suitable speeds are below8000 rpm, preferably below 6000 rpm, and in a specific embodimentdescribed below the speed is about 3000 rpm.

The method may be performed at ambient temperature, which is convenientfor laboratory and industrial purposes, and may also be performed atbelow ambient temperature improves the stability of the plasmid DNAduring the encapsulation procedures. The temperature of the method maybe reduced to below 20° C., below 10° C. or even below 5° C. In anembodiment of the invention, the method is carried out at below ambienttemperature using a reduced amount of microparticle precursor comparedto the amount used at ambient temperature.

The parameters of the method are thus chosen to promote formation ofmicroparticles of 10 μm diameter or less and to promote incorporation ofDNA into microparticles, and to avoid damage to the DNA such that theDNA can not be expressed in the recipient.

For any particular choice of polymer and DNA variations in the methodmay be necessary to obtain best results. The efficiency of a method canbe assessed by transformation or transfection assays. In thetransformation assay used by the inventors, DNA is recovered frommicroparticles by dissolution with organic solvent, quantitated and usedto transform bacteria—ampicillin selection determines successfultransformants. In the transfection assay, recovered DNA is used totransfect eukaryotic cells in culture, which culture is then assayed forpresence of the antigen or gene therapy product. These assays havedemonstrated that DNA recovered from microparticles produced by themethod of the invention can retain 50-60% and up to 80% of the activityof the original DNA, indicating high efficiency of incorporation offunctional DNA into microparticles.

In a further embodiment of the invention there is provided a method ofmaking a pharmaceutical composition comprising preparing a DNA constructfor expression of a coding sequence within the construct, and formingaround the construct a polymer particle of size between 0.01 μm and 30μm, wherein the construct remains capable of inducing expression of thecoding sequence. In use, when the construct is separated from theparticle, it induces expression of the coding sequence. The particle ispreferably formed by emulsifying a solution of a polymer plus DNA plusalcohol.

The method of the invention is adapted to produce pharmaceuticalcompositions of the first aspect of the invention. The steps of themethod are adapted so that, in a resultant composition which containsmany DNA containing polymer particles, a useful proportion of particlescontain active DNA, i.e. DNA that has not been damaged by the methodsuch that its ability to induce expression of its coding sequence islost. DNA activity is measured as a percentage of activity prior to theparticle forming step.

An acceptable level of DNA biological activity is at least 10% andpreferably at least 25%, though for particularly fragile DNA a lowerpercentage may be acceptable so long as, in use, a therapeutic effect isdemonstrated by the composition.

In a specific embodiment of the invention, a composition is made bypreparing a solution of a plasmid of double stranded, supercoiled DNAcomprising a coding sequence and a eukaryotic promoter. Separately, apolymer solution is prepared. The two solutions are mixed together andemulsified at a speed between 1000 and 4000 rpm. A solution of astabilizing agent is then added and the new mixture emulsified at aspeed between 1000 and 4000 rpm. After centrifugation and resuspensionof particles the DNA within retains 25% of its activity.

A third aspect of the invention provides a pharmaceutical compositioncomprising polymer—encapsulated DNA and having a reduced water content,such as less than 5% by weight. This composition is suitable for longterm storage while retaining the ability of the DNA, upon administrationto a recipient, to induce expression of a coding sequence within saidDNA.

A method of preparing a pharmaceutical composition for storage, is todry, such as by freeze drying, a pharmaceutical composition according tothe first aspect of the invention. It is preferred that the driedcomposition has a water content of less than 5%, though the precisewater content will be determined by the period of drying used.

A fourth aspect of the invention provides a method of vaccinationcomprising administering a vaccine according to the first aspect of theinvention. Vaccination can thus be obtained by eliciting antibodies tothe immunogen expressed from the gene coding sequence. As will beappreciated, the immunogen can be a component of a virus or bacterium orother pathogenic microorganism, or can be

EXAMPLES

an analogue of said immunogen such that antibodies against the analogueare effective against the pathogen itself.

In a specific embodiment of the invention described in an example below,the particle material is PLG. The size of particles produced by themethod of the invention are generally in the range of 0.01-30 μm,preferably 1-10 μm. Other suitable polymer formulations forDNA—containing particles according to the present invention includepoly-lactic acid, poly-hydroxybutyrate, poly hydroxyvalerate, poly(hydroxybutrate/valerate), ethyl cellulose, dextran, polysaccharides,polyalkylcyanoacrylate, poly-methyl-methacrylate, poly(e-caprolactone)and mixtures of all of these components.

In use of a specific embodiment of the invention, described in anexample below, a preparation of microparticles according to theinvention comprises DNA coding for the protein luciferase. As will beappreciated by a person of skill in the art, a wide range of DNAsequences and constructs are suitable for use in this invention. Inparticular, the invention can be practised incorporating a wide range ofplasmid vectors already well known and characterised in the art.Typically, a plasmid vector used in this invention will include a cDNAthat codes for the desired gene product. The selection of additionalcomponents for the DNA sequence, such as promoters, reporter genes andtranscription termination sequences can be made by a person of skill inthe art according to common general knowledge concerning construction ofknown plasmid vectors.

The preferred administration route for compositions of the invention isthe oral route, meaning that compositions of the invention shouldpreferably be designed to avoid significant degradation while passingthrough the stomach with its high acid levels. It is known that uptakeof microparticles of less than 10 μm in size occurs, inter alia, in theM cells of the intestine, and thus inclusion of DNA containing particlesin this size range can be advantageous in promoting uptake at thisintestinal location. Other modifications to the nature and character andcomponents of the polymer can be made within the concept of theinvention.

There now follows description of specific embodiments of the invention,accompanied by figures in which:

FIG. 1 is a schematic diagram of the components of a protein-expressingplasmid suitable for incorporation into a DNA-containing particleaccording to the invention;

FIG. 2 illustrates the results of example 3, namely induction ofluciferase-specific serum antibodies by PLG-encapsulated plasmid DNA,and in which the left column indicates antibody titre after 3 weeks andthe right column indicates antibody titre after 6 weeks (i.m. representsintra muscular, i.p. represents intra peritoneal, the bottom portion ofeach column represents IgG levels, the top portion represents IgMlevels);

FIG. 3 shows dose-response data for injected and oral doses ofencapsulated DNA, with the titre of IgG, IgM and IgA in each case.

FIG. 4 shows: A—agarose gel electrophoresis of plasmid DNA followinghomogenisation in a Silverson mixer at 2000 and 8000 rpm for 0-300seconds; and B—agarose gel electrophoresis of DNA before and afterencapsulation;

FIG. 5 shows stool anti-luciferase IgA response to DNA within PLGmicroparticles.

FIG. 6 shows the results of oral administration of PLG-encapsulated DNAexpressing measles virus N protein; and

FIG. 7 shows the results of oral administration of PLG-encapsulated DNAexpressing rotavirus VP6 gene: A—faecal rotavirus—specific IgA response,B—rotavirus shedding after challenge of orally immunized mice.

Example 1

Method for Encapsulation of Plasmid DNA in PLG Microparticles Equipment:

1) Silverson Laboratory mixer with ¾″ probe fitted with emulsor screen.

2) High speed centrifuge.

3) Normal laboratory glassware, beakers, measuring cylinders, stirrersetc.

Reagents:

1) Poly(lactide-co-glycolide) (PLG) solution—500 mgs in 3 mldichloromethane.

2) Plasmid DNA (12 mg/ml in water).

3) Polyvinyl alcohol (PVA) solution (8% w/v in water).

4) Absolute ethanol.

5) TEN buffer (10 mM tris pH 8.0+1 mM EDTA+50 mM NaCl).

Method:

1) Mix 200 μl plasmid DNA solution with 250 μl of TEN and add 150 μlethanol with stirring. Mix well.

2) Add this mixture to 3 ml PLG solution and emulsify in the Silversonmixer at 3000 rpm for 2 min.

3) Add this emulsion to 100 ml PVA and emulsify at 3000 rpm for 2 min.

4) Add the double emulsion to 1 litre of water and stir vigorously for 1min.

5) Distribute the suspension of microparticles in centrifuge containersand centrifuge at 10,000×g_(av) for 30 mins.

6) Resuspend the microparticle pellet in 25 ml of water and homogenisewith a hand homogeniser with large clearance (0.5 mm) to make ahomogeneous suspension. Dilute with 200 ml of water and recentrifuge asabove.

7) Repeat step 6 three times.

8) Resuspend the microparticle pellet in 25 ml of water as above,transfer to a vessel suitable for freeze drying, shell freeze in anisopropanol/dry ice mixture and lyophilise for 48 h.

In this method, steps 1-3 are carried out at ambient temperature. DNAwas incorporated into the microparticles with an efficiency of about25%.

Example 2

Plasmids for the Expression of Proteins after in vivo Delivery

Suitable plasmids for use in microparticles according to the inventionconsist of the following components (see FIG. 1):

1. Plasmid Backbone The plasmid backbone has an origin of replicationand an antibiotic resistance gene or other selectable marker to allowmaintenance of the plasmid in its bacterial host. Backbones providing ahigh copy number will facilitate production of plasmid DNA. An examplewould be from the pUC plasmid vectors.

2. Transcriptional Promoter Sequence Expression of the desired proteinwill be driven by a, typically eukaryotic, transcriptional promoterinitiating the synthesis of mRNA. Generally, strong promotersfunctioning in a wide variety of tissue types and animal species are tobe used, e.g. the human cytomegalovirus immediate early (hCMV IE)promoter. However, particularly for gene therapy applications, atissue—or cell type—specific promoter may be more appropriate.

3. Coding Sequence The coding sequence contains the DNA sequenceencoding the protein of interest. It contains the translational startcodon ATG in sequence context favourable for initiation of proteinsynthesis. The coding sequence ends with a translational terminationcodon. Proteins to be expressed include a) reporter enzymes (e.g.luciferase, β-galactosidase); b) components of pathogenic microorganismscapable of inducing protective immune responses (e.g. the NS1 protein oftick-borne encephalitis virus, the N, H or F proteins of measles virus,the gp 120 protein of human immunodeficiency virus 1); c) enzymes orother proteins intended for the treatment of genetic disease (e.g.glucocerebrosidase for the treatment of Gaucher's disease).

4. Transcription Termination Sequence Improved expression levels areobtained under some circumstances when sequences causing termination ofmRNA transcription are incorporated downstream of the coding sequence.These sequences frequently also contain signals causing the addition ofa poly A tail to the mRNA transcript. Sequences which can be used inthis role can be derived from the hCMV major immediate early proteingene or from SV40 viral DNA or elsewhere.

Example 3

We have constructed a plasmid encoding the insect protein luciferase,under the transcriptional control of the human cytomegalovirus immediateearly (hCMV IE) promoter, and demonstrated luciferase activity in cellstransfected in vitro.

We encapsulated purified plasmid DNA in PLG microparticles around 2 μmin size with moderate (about 25%) efficiency using the protocol ofExample 1. Agarose gel electrophoresis indicates that a proportion ofthe initially closed circular supercoiled DNA undergoes conversion to amore slowly migrating form, probably relaxed circles, as a result ofshear stresses in the encapsulation process. The encapsulated DNA wasreleased from the particles and shown to retain a significant fractionof its in vivo biological activity in assays of bacterial transformationby electroporation, and luciferase expression after transfection intocultured cells.

Microencapsulated DNA (50 μg) was administered to mice byintraperitoneal (i.p.) injection and orally. Control animals receivedunencapsulated DNA by the same routes, and as a positive control bystandard intramuscular (i.m.) injection. Luciferase-specific serumantibodies were analyzed by ELISA three and six weeks after DNAadministration. Results are presented in FIG. 2.

As shown in FIG. 2, modest specific IgG and IgM responses were seenafter i.m. injection, as might be expected. Encapsulated DNA evokedstrong IgG and IgM responses after i.p. injection, while unencapsulatedDNA gave much weaker responses. Similarly, orally administeredencapsulated DNA evoked a good IgG response which was not matched byunencapsulated DNA. The IgG and IgM antibody responses indicate thatluciferase expression and presentation to the immune system occur afteradministration of plasmid DNA encapsulated in PLG microparticles withefficiencies exceeding that seen with the standard i.m. route, andexceeding that seen in comparative administration of unencapsulated DNA.

Example 4

In further experiments, microencapsulated DNA, made by the method ofExample 1, in a range of doses (1-50 μg DNA) was administered to groupsof outbred mice by intra-peritoneal (i.p.) injection or orally.Luciferase-specific serum antibodies of IgG, IgM and IgA classes wereanalysed by ELISA at 3, 6 and 9 weeks after DNA administration.

In FIG. 3, it can be seen that i.p. injection of PLG-encapsulated DNAevoked good IgG and IgM responses, and a modest IgA response. Orallyadministered encapsulated DNA evoked good responses in all threeantibody classes. There is a trend for the antibody titres to increasewith time after DNA administration, and the responses are alsodose-related to a greater or lesser extent. It is apparent thatquantities of DNA as low as 1 μg are able to evoke significantresponses, especially at longer times after administration. Theseantibody responses again confirm that luciferase expression occurs afteradministration of plasmid DNA encapsulated in PLG microparticles, eitherby i.p. injection or orally. They also demonstrate that antigen ispresented to the immune system by these means in such a fashion as toevoke IgG, IgM and IgA classes of antibody.

Example 5

We examined the effect of high-speed homogenisation steps, used togenerate the required water-oil-water emulsions which are intermediatesin the encapsulation process, on the physical integrity and biologicalfunction of plasmid DNA.

In initial experiments, supercoiled plasmid DNA was adjusted toconcentrations and volumes similar to those to be used inmicroencapsulation experiments, and homogenised with a Silversonlaboratory homogeniser. Samples were removed at intervals from 0 to 300sec for analysis by agarose gel electrophoresis (FIG. 4A). Such ananalytical procedure is capable of distinguishing between supercoiled(sc) DNA, open circular (oc) DNA, where a single strand has been nicked,and linear (I) DNA, where both strands have been cut at adjacent points(see, for example, FIG. 2C in Garner and Chrambach 1992. Resolution ofcircular, nicked and linear DNA, 4.4 kb in length, by electrophoresis inpolyacrylamide solutions. Electrophoresis 13, 176-178). It is clear thatexposure to such conditions for periods as short as 10 sec results inconversion from sc to oc form. At 8000 rpm, further conversion to thelinear form and eventually more extensive degradation occur. However, atthe reduced speed of 2000 rpm the oc form of DNA is relatively stableover the time period typically required for formation of the emulsionintermediates involved in PLG encapsulation. These studies thus showthat plasmid DNA is vulnerable to shear-induced damage, and carefulattention is required to the precise conditions to obtain encapsulationof minimally altered DNA.

From this basis, we have developed conditions for the encapsulation ofpurified plasmid DNA in PLG microparticles around 2 μm in size withmoderate (about 25%) efficiency. Agarose gel electrophoresis (FIG. 4B)indicates that the initially closed circular supercoiled DNA undergoesconversion to the oc form, as a result of shear stresses in theencapsulation process. Biological activity of DNA released frommicroparticles has been assessed in assays of bacterial transformationby electroporation, and luciferase expression after transfection intocultured cells. DNA released from the particles retains a significantfraction (about 25%) of its in vitro activity in both these assays.

Example 6

PLG-encapsulated DNA coding for luciferase, made by the method ofExample 3, is also able to evoke a mucosal immune response to theexpressed protein. Levels of IgG, IgM and IgA antibodies specific forluciferase were assessed by ELISA in stool samples from mice whichreceived i.p. or oral doses of 1, 5, 20 or 50 μg of PLG-encapsulatedDNA. No significant levels of IgG or IgM antibodies were found in stoolsamples from any group of mice. Rather limited IgA responses were seenin the i.p.-injected mice; however, oral administration resulted insignificant levels of luciferase-specific IgA antibodies in the stoolsamples (FIG. 5). These reached extraordinarily high levels in thosemice which received 50 μg PLG-encapsulated DNA. These results indicatethat oral administration of a single dose of PLG-encapsulated plasmidDNA is capable of evoking a mucosal, as well as a systemic antibodyresponse. This may be a useful attribute of a PLG-encapsulated DNAvaccine in applications where protection against infection at mucosalsurfaces is desirable, as for measles or AIDS.

Example 7

We have exploited a plasmid expressing the measles virus (MV)nucleocapsid protein (N) to extend our observations that the oraladministration of encapsulated plasmid DNA expressing luciferase iscapable of eliciting a systemic antibody response. The N-expressingconstruct is identical to that expressing luciferase (described inexample 3), except for the replacement of the coding sequence with theEdmonston strain MV N coding sequence. The purified plasmid DNA wasPLG-encapsulated (using the method as described in example 1).

Inbred C3H mice were immunized with two doses suspended in 0.1 M Sodiumbicarbonate administered by oral gavage, 13 days apart; each dosecontained 50 μg DNA. Control groups of mice received PBS alone or PLGparticles containing plasmid vector DNA containing no coding sequence.Mice were bled at intervals and serum levels of IgG specific for MV Ndetermined by ELISA, using recombinant MV N expressed in insect cells asantigen. As shown in FIG. 6A, immunization with PLG-encapsulated DNAexpressing MV N resulted in significant levels of N-specific antibody;results shown are mean absorbances in 1/100 diluted sera taken 53 daysafter the second DNA administration. There seems to be a considerabledegree of variability in the response of individual mice to DNAimmunization in these experiments (see FIG. 6B), but very high levels ofantibody (reciprocal titres exceeding 10⁴, determined in follow-upexperiments) are present in some animals. These results demonstrate thatoral delivery of PLG-encapsulated DNA is an effective method forinducing an immune response against an important pathogen.

Example 8

A Further Method for Encapsulation of Plasmid DNA in PLG MicroparticlesEquipment:

1) Silverson Laboratory mixer with ¾″ probe fitted with emulsor screen.

2) High speed centrifuge.

3) Normal laboratory glassware, beakers, measuring cylinders, stirrersetc.

Reagents:

1) Poly(lactide-co-glycolide) (PLG) solution—500 mgs in 3 mldichloromethane.

2) Plasmid DNA (>10 mg/ml in TE buffer).

3) Polyvinyl alcohol (PVA) solution (8% w/v in water).

4) Absolute ethanol.

5) TE buffer (10 mM tris pH 8.0+1 mM EDTA+50 mM NaCl).

Method:

1) Mix 450 μl plasmid DNA solution with 150 μl ethanol with stirring.Mix well.

2) Add this mixture to 3 ml PLG solution and emulsify in the Silversonmixer at 2000 rpm for 2½ min.

3) Add this emulsion to 100 ml PVA and emulsify at 2000 rpm for 2½ min.

4) Add the double emulsion to 1 litre of water and stir vigorously for 1min.

5) Distribute the suspension of microparticles in centrifuge containersand centrifuge at 10,000×g_(av) for 30 mins.

6) Resuspend the microparticle pellet in 25 ml of water and homogenisewith a hand homogeniser with large clearance (0.5 mm) to make ahomogeneous suspension. Dilute with 200 ml of water and recentrifuge asabove.

7) Repeat steps 5 and 6 four times.

8) Resuspend the microparticle pellet in 25 ml of water as above,transfer to a vessel suitable for freeze drying, shell freeze in anisopropanol/dry ice mixture and lyophilise for 48 h.

In this method, steps 1-3 are carried out at ambient temperature. Theefficiency was improved compared to example 1, up to 30-40% efficiency.

Example 9

A Further Method for Encapsulation of Plasmid DNA in PLG MicroparticlesEquipment:

1) Silverson Laboratory mixer with ¾″ probe fitted with emulsor screen.

2) High speed centrifuge.

3) Normal laboratory glassware, beakers, measuring cylinders, stirrersetc.

Reagents:

1) Poly(lactide-co-glycolide) (PLG) solution—400 mgs in 3 mldichloromethane.

2) Plasmid DNA (>10 mg/ml in TE buffer).

3) Polyvinyl alcohol (PVA) solution (8% w/v in water).

4) Absolute ethanol.

5) TE buffer (10 mM tris pH 8.0+1 mM EDTA.

Method:

1) Mix 450 μl plasmid DNA solution with 150 μl ethanol with stirring.Mix well.

2) Add this mixture to 3 ml PLG solution and emulsify in the Silversonmixer at 2000 rpm for 2½ min.

3) Add this emulsion to 100 ml PVA and emulsify at 2000 rpm for 2½ min.

4) Add the double emulsion to 1 litre of water and stir vigorously for 1min.

5) Distribute the suspension of microparticles in centrifuge containersand centrifuge at 10,000×g_(av) for 30 mins.

6) Resuspend the microparticle pellet in 25 ml of water and homogenisewith a hand homogeniser with large clearance (0.5 mm) to make ahomogeneous suspension. Dilute with 200 ml of water and recentrifuge asabove.

7) Repeat steps 5 and 6 four times.

8) Resuspend the microparticle pellet in 25 ml of water as above,transfer to a vessel suitable for freeze drying, shell freeze andlyophilise for 48 h.

In this method, steps 1-3 are carried out at 4° C. The efficiency ofincorporation of DNA into microparticles was 50-60%.

Example 10

Plasmid DNA (pCMVIA/VP6) expressing the VP6 gene of murine rotavirus(epizootic diarrhoea of infant mice (EDIM) virus) was constructed at theUniversity of Massachusetts Medical Center. The gene encoding VP6 wasinserted into a vector (pJW4303) downstream of sequences of theimmediate early transcriptional promoter and intron A of humancytomegalovirus and a tPA-derived secretory signal sequence. The genewas followed by transcriptional termination and polyadenylationsequences derived from the bovine growth hormone gene. The purifiedplasmid DNA was PLG-encapsulated using the method described in example1.

Inbred Balb/c mice were immunized by oral administration with a dosecontaining 50 micrograms VP6-expressing DNA encapsulated in PLGmicroparticles. A control group of mice received a similar dose ofencapsulated vector DNA. Mice were examined for intestinalrotavirus-specific IgA at fortnightly intervals by ELISA, using EDIMvirus as antigen. Nine weeks after immunisation, the animals werechallenged with EDIM virus, and monitored for excretion of virus instool using a monoclonal antibody-based ELISA.

As shown in FIG. 7A, immunisation with PLG-encapsulated DNA expressingVP6 resulted in significant levels of rotavirus-specific intestinal IgAantibody when compared with the control animals. This is particularlynoteworthy, since other routes of administration of VP6-expressingplasmid DNA do not elicit detectable levels of intestinal virus-specificIgA before virus challenge. After challenge with EDIM virus, there was areduction in rotavirus shedding in mice which had receivedPLG-encapsulated VP6-expressing plasmid DNA, compared with the controlgroup (FIG. 7B).

These results demonstrate that orally administered PLG-encapsulatedplasmid DNA is capable of inducing a) a specific intestinal IgAresponse, and b) protection against virus challenge, as manifested in areduction in virus shedding.

The compositions and methods of the invention have application in slowrelease systems for delivery of DNA vaccines; prolonged expression ofimmunogen potentially results in efficient single dose priming andboosting, with consequent efficient induction of long term memoryresponses. Another application is in a vehicle for the oral delivery ofvaccines; simple and acceptable means for vaccine administration arelikely to improve vaccine uptake rates; in addition, freeze-driedencapsulated plasmid DNA is likely to be very stable and insensitive toenvironmental conditions. A further application is in a slow releasesystem for gene therapy; prolonged release of DNA and subsequentexpression potentially reduces the need for repeated treatment.

What is claimed is:
 1. A synthetic composition comprising a polymermicrocapsule and DNA, wherein the DNA (a) is inside the microcapsule,(b) comprises a promoter linked to a sequence coding for an immunogen,and (c) exhibits at least 25% of its pre-encapsulation activity, asassayed by transformation of competent bacteria; and wherein themicrocapsule is 10 μm or less in diameter.
 2. The method of claim 1wherein the DNA is plasmid DNA.
 3. The method of claim 1 wherein the DNAcomprises a sequence promoting transcription of the sequence coding forthe immunogen.
 4. The method of claim 1, wherein the compositioncomprises a plurality of said DNA-containing microcapsules wherein atleast 50% of said microcapsules are in the size range 1 μm to 10 μm. 5.The method of claim 1, wherein the immunogen induces an immune responsethat comprises production of antibodies specific to the immunogen. 6.The method of claim 5 wherein the immune response comprises productionof IgA antibodies.
 7. The method of claim 1, wherein the compositioncomprises a pharmaceutically acceptable carrier.
 8. The method of claim7 wherein said immunogen is an immunogenic component of an organismselected from the group consisting of a virus and a bacterium.
 9. Themethod of claim 8 wherein the immunogen is a viral protein.
 10. Themethod of claim 1, wherein the polymer has a solubility in methylenechloride of at least 100 mg/ml.
 11. The method of claim 1, wherein themicrocapsule comprises supercoiled DNA.
 12. The method of claim 1wherein said immunogen elicits a T cell response.
 13. The method ofclaim 12 wherein said T cell response is a cytotoxic T cell (CTL)response.
 14. The method of claim 1 wherein said polymer comprisespoly(lactide-co-glycolide)(PLG).
 15. The method of claim 1, wherein theDNA in said composition retains 50-60% of the pre-encapsulationactivity.
 16. The method of claim 1, wherein the DNA in said compositionretains up to 80% of the pry-encapsulation activity.
 17. The method ofclaim 1, further comprising formulating the composition in the form of adry powder.
 18. The method of claim 1, wherein said polymer consists ofPLG.
 19. The method of claim 1, where in the polymer is selected fromthe group consisting of a lactide-containing polymer, aglycolide-containing polymer, and a polymer containing lactide andglycolide.
 20. The method of claim 1, wherein the emulsifying speed isbelow 6000 rpm.
 21. The method of claim 1, wherein the emulsifying speedis below 3000 rpm.
 22. The method of claim 1, wherein the emulsifyingspeed is between 1000 and 4000 rpm.
 23. A method of administering acomposition to a mammal, comprising: preparing a composition accordingto the method of claim 7; and administering the composition to a mammalin a manner effective to elicit antibodies against the immunogen.
 24. Amethod of inducing production of an antibody in an animal, comprising:preparing a composition according to the method of claim 5; andadministering to said animal an effective amount of the composition. 25.A method of administering a nucleic acid to an animal, comprising:preparing a composition according to the method of claim 1; andintroducing the composition into the animal.
 26. The method of claim 25,wherein the DNA in said composition retains 50-60% of thepre-encapsulation activity.
 27. The method of claim 25, wherein the DNAin said composition retains up to 80% of the pre-encapsulation activity.28. A method of eliciting production of IgA antibodies specific for animmunogen, the method comprising: preparing a composition according tothe method of claim 1; and orally administering the composition to amammal.